The peripheral-type benzodiazepine receptor (PBR) was originally discovered because it binds the benzodiazepine diazepam with relatively high affinity (Papadopoulos, V. 1993, Endocr. Rev. 14:222–240). Benzodiazepines are among the most highly prescribed drugs due to their pharmacological actions in relieving anxiety mediated through modulating the activity of γ-aminobutyric acid receptors in the central nervous system (Costa, E. and Guidotti, A. 1979, Annu. Rev. Pharmacol. Toxicol. 19:531–545). PBR is another class of binding sites for benzodiazepines distinct from the aforementioned neurotransmitter receptors. Further studies demonstrated that in addition to benzodiazepines, PBR binds other classes of organic compounds with high affinity (Papadopoulos, 1993, supra). PBR, although present in all tissues examined, was found to be particularly high in steroid producing tissues, where it was primarily localized in the outer mitochondrial membrane (OMM) (Anholt, R. R. H. et al. 1986, J. Biol. Chem. 261:576–583). An 18 kDa isoquinoline-binding protein was identified as PBR, cloned and expressed (Papadopoulos, V. 1998, Proc Soc. Exp. Biol. Med. 217:130–142). It was then demonstrated that PBR is a functional component of the steroidogenic machinery (Papadopoulos, 1998, supra; Papadopoulos V. et al. 1990, J. Biol. Chem. 265:3772–3779) mediating cholesterol delivery from the outer to the inner mitochondrial membrane (Krueger, K. E. and Papadopoulos, V. 1990, J. Biol. Chem. 265:15015–15022). Further studies demonstrated that pharmacologically induced reduction of adrenal PBR levels in vivo resulted in decreased circulating glucocorticoid levels (Papadopoulos, V. 1998, supra) In addition, targeted disruption of the PBR gene in Leydig cells resulted in the arrest of cholesterol transport into mitochondria and steroid formation; transfection of the mutant cells with a PBR cDNA rescued steroidogenesis (Papadopoulos, V. et al. 1997, J. Biol. Chem. 272:32129–32135).
PBR is extremely abundant in steroidogenic cells and found primarily on outer mitochondrial membranes (Anholt, R. et al. 1986, J. Biol. Chem. 261:576–583). PBR is thought to be associated with a multimeric complex composed of the 18-kDa isoquinoline-binding protein and the 34-kDa pore-forming voltage-dependent anion channel protein, preferentially located on the outer/inner mitochondrial membrane contact sites (McEnery, M. W. et al. Proc. Natl. Acad. Sci. U.S.A. 89:3170–3174; Garnier, M. et al. 1994, Mol. Pharmacol. 45:201–211; Papadopoulos, V. et al. 1994, Mol. Cel. Endocr. 104:R5–R9). Drug ligands of PBR, upon binding to the receptor, simulate steroid synthesis in steroidogenic cells in vitro (Papadopoulos, V. et al. 1990, J. Biol. Chem. 265:3772–3779; Ritta, M. N. et al. 1989, Neuroendocrinology 49: 262–266; Barnea, E. R. et al. 1989, Mol. Cell. Endocr. 64:155–159; Amsterdam, A. and Suh, B. S. 1991, Endocrinology 128:503–510; Yanagibashi, K. et al. 1989, J. Biochem. (Tokyo) 106: 1026–1029). Likewise, in vivo studies showed that high affinity PBR ligands increase steroid plasma levels in hypophysectomized rats (Amri, H. et al. 1996, Endocrinology 137:5707–5718). Further in vitro studies on isolated mitochondria provided evidence that PBR ligands, drug ligands, or the endogenous PBR ligand, the polypeptide diazepam-binding inhibitor (BDI) (Papadopoulos, V. et al. 1997, Steroids 62:21–28), stimulate pregnenolone formation by increasing the rate of cholesterol transfer from the outer to the inner mitochondrial membrane (Krueger, K. E. and Papadopoulos, V. 1990, J. Biol. Chem. 265:15015–15022; Yanagibashi, K. et al. 1988, Endocrinology 123: 2075–2082; Besman, M. J. et al. 1989, Proc. Natl. Acad. Sci. U.S.A. 86: 4897–4901; Papadopoulos, V. et al. 1991, Endocrinology 129: 1481–1488).
Based on the amino acid sequence of the 18-kDa PBR, a three dimensional model was developed (Papadopoulos, V. 1996, In: The Leydig Cell. Payne, A. H. et al. (eds) Cache River Press, IL, pp 596–628). This model was shown to accomodate a cholesterol molecule and function as a channel, supporting the role of PBR in cholesterol transport. Recently we demonstrated the role of PBR in steroidogenesis by generating PBR negative cells by homologous recombination (Papadopoulos, V. et al. 1997, J. Biol. Chem. 272:32129–32135) that failed to produce steroids. However, addition of the hydrosoluble analogue of cholesterol, 22R-hydroxycholesterol, recovered steroid production by these cells, indicating that the cholesterol transport mechanism was impaired. Further cholesterol transport experiments in bacteria expressing the 18-kDa PBR protein provided definitive evidence for a function as a cholesterol channel/transporter (Li and Papadopoulos, 1998, Endocrinology 139, 4991–4997).
Studies in a number of tumors such as rat brain containing glioma tumors (Richfield, E. K. et al. 1988, Neurology 38:1255–1262), colonic adenocarcinoma and ovarian carcinoma (Katz, Y. et al. 1988, Eur. J. Pharmacol. 148: 483–484 and Katz, Y. et al. 1990, Clinical Sci. 78:155–158) have shown an abundance of peripheral-type benzodiazepine receptors (PBR) compared to normal tissue. All documents cited herein infra and supra are hereby incorporated in their entirety by reference thereto. Moreover, a 12-fold increase in PBR density relative to normal parenchyma, was found in human brain glioma or astrocytoma (Cornu, P. et al. 1992, Acta Neurochir. 119:146–152). The authors suggested that PBR densities may reflect the proliferative activity of the receptor in these tissues. Recently, the involvement of PBR in cell proliferation was further shown (Neary, J. T. et al. 1995, Brain Research 675:27–30; Miettinen, H. et al. 1995, Cancer Research 55:2691–2695), and its expression in human astrocytic tumors was found to be associated with tumor malignancy and proliferative index (Miettinen, H. et al. supra; Alho, H. 1994, Cell Growth Different. 5:1005–1014). Characterization of PBR in human breast cancer biopsies, led to the discovery that the invasive and metastatic ability of human breast tumor cells is proportional to the level of PBR expressed, and correlates with the subcellular localization of PBR in these cells in that PBR is found primarily in the nucleus in aggressive tumor cells whereas PBR is found primarily in the cytoplasm of invasive but non-aggressive cells. These changes in PBR expression can be used as a tool for detection, diagnosis, prevention and treatment in breast cancer patients, in particular, and in aggressive solid tumors in general.
Since both PBR and its endogenous ligand, the polypeptide diazepam binding inhibitor, are constitutively expressed in steroidogenic cells, the regulation of PBR function by hormones may be due to its association with other proteins. This interaction may result in the initiation of steroid biosynthesis. Therefore, there is a need to identify proteins which associate with PBR and may modulate PBR function.